This project is designed to identify epitopes on HIV proteins towards monoclonal antibodies using a combination of proteolytic footprinting of the protein affinity bound to an immobilized antibody. Identification of peptide sequences that remain bound to the antibody after proteolysis is based on matrix-assisted laser desorption mass spectrometry. This methodology permits the investigation of successive proteolytic enzymes so that the epitope can be narrowly defined. This technique should also be applicable to the investigation of discontinuous epitopes. The HIV protein chosen for initial investigation is p24, a core structural protein. P24 is a major target for antibody response and is one of the first antibodies to appear during HIV infection. We have used either an aliquot of p24 from the NIH OAR ARRRP or recombinant p26 expressed from E. coli in our lab. These materials bound to several commercial monoclonal antibodies under native conditions. After treatment of the affinity-bound antibodies with Lys-C, three fragments were observed to be still affinity bound (residues 71-131, 132- 140, and 171-182). Further treatment with trypsin resulted in loss of residues 132-140 and truncation of the 71-131 fragment to residues 83-97. Residues 171-182 were still bound even though the sequence contains an arginine at residue 173. Preliminary experiments with Aminopeptidase M indicate that residue 83 can be removed. These results indicate that three sequences are involved in the binding of the epitope to the MAb. These residues may, therefore, represent a discontinuous epitope.